Agricultural compositions containing bacteria

ABSTRACT

An agriculturally effective active ingredient is applied to plant foliage before, after, or simultaneously with an enhancer component containing a substantially pure bacterial culture, suspension, spores, or cells of a bacteria selected from the genus Bacillus or a soil bacteria.

This application is a national phase application claiming priority to PCT/US96/19116, filed on Nov. 29, 1996, and is a continuation in part of U.S. patent application Ser. No. 08/453,683 filed on May 30, 1995, now U.S. Pat. No. 5,650,372.

FIELD OF THE INVENTION

The invention relates to the treatment of plants by a composition containing an agriculturally effective active ingredient and an enhancer additive containing a substantially pure culture of bacteria selected from the genus Bacillus or a soil bacteria. The added culture may be in the form of cell, spores, or suspensions.

BACKGROUND OF THE INVENTION

Agricultural chemical manufacturers are always looking for ways to improve the efficacy of active ingredients used on plants. This is particularly true where the applied material is a plant growth regulator (growth stunting or growth enhancing) herbicide, or systemic agent (e.g., insecticide or fungicide). Transport mechanisms into the plant and translocation among the various plant tissues is important and, in some instances, may be the primary factor determining the efficacy of the applied ingredient. For some active ingredients, an improvement in the transports mechanism could translate into improved performance at existing application rates, the need for less active ingredient, or the ability to treat new species that were previously resistant to the active ingredient.

Mepiquat chloride is an active ingredient where plant uptake and transport is important. Mepiquat chloride (N,N-dimethylpiperidinium chloride) is used annually as the active ingredient for stunting vegetative cotton plant growth and increasing fruit retention on millions of acres of cotton. Mepiquat chloride also has some uses on potatoes, sweet potatoes, peanuts, grapes, corn, wheat, citrus, tomatoes, and onions.

Mepiquat chloride has the effect on cotton plants of stunting vegetative growth thereby forcing the plant to redirect its energies into fruit (cotton boll) production. With appropriate application of mepiquat chloride to plants that are beginning to exhibit excessive vegetative growth, cotton plant yields can be maintained or increased without harm to the plant. The growth stunting effects are particularly desired when the cop is grown in fertile soil or after weather conditions that favor plant growth rather than fruit production.

Cotton plants have a predictable life cycle and growth period. Cotton plants emerge 7-10 days after the seeds are planted in a furrow. The cotton plant exhibits growth of a root system and the extension of plant height through stem and branch growth in a pattern referred to as “vegetative growth” until about the 4th-8th node. Thereafter, the plant produces a reproductive branch (the “1st fruiting site”), and all subsequent branches are reproductive. Cotton growers attempt to control the growth of the plant to ensure that the ratio of vegetative growth to reproductive growth (boll production) favors the desired range of reproductive growth.

Cotton growers generally prefer to see about 2 inches (5 cm) between main stem nodes. This ratio represents a balance between too much reproductive growth (boll production) which can cause the plant growth to outpace the rate of vegetative growth and terminate before the yield is maximized, and too much vegetative growth which reduces the number of mature bolls.

Cotton plants that have directed the majority of the available plant energy to vegetative growth are referred to as “rank” cotton and produce less bolls which mature later and are vulnerable to weather extremes for longer periods of time. Cotton that exhibits signs of going rank are readily visible by abnormal plant height relative to the boll loads and number of reproductive main stem nodes. Mepiquat chloride is used to stop cotton from going rank by modifying the cotton plant's growth characteristics.

The branches off the main stem generally always extend from alternating sides of the stem. Each branch site is called a “node” with 5-7 nodes being formed above the cotyledon leaves before the first fruit bearing branch with true leaves is formed. Node counting starts at the bottom of the plant and extends up the main stem. The “internode length” is the distance between branch sites with a new node being formed roughly every three days. For purposes of measurement and comparison, the number of nodes and internode length above node 8 are generally used to eliminate interplant fruiting node variations because fruit bearing branches will necessarily have been formed by node 8. The counting of fruiting nodes thus conventionally starts from the first reproductive node, usually no. 7 or no. 8.

Fruiting sites in cotton are referred to as “squares.” Each fruit bearing branch will form 1-6, normally about 3, fruiting sites (“squares”) with approximately six days between square formations on each branch. New squares and the beginning of reproductive growth in cotton plants are referred to as “pinhead” squares due to their barely visible size. After about 5-10 days, the square has grown to about the size of a match head and is a period in the plant cycle referred to as a “match head square.” The match head square continues to grow to about the size of an average adult fingernail before blooming (“early bloom”). Three days later, a boll has formed beneath the bloom. Roughly thirty days after early bloom, the product boll is fully mature and ready for harvest. Overall, about 80% of the total cotton yield is set within the first 3 weeks after early bloom and 95% of the total yield is set within 5 weeks of early bloom.

Generally, mepiquat chloride is applied to cotton plants in one of two ways. The method used until about 1986 was a single application of 8-16 ounces per acre of a 4.2 wt % solution at early bloom. This type of single treatments did control plant height although it was noticed that plant yields were occasionally reduced particularly if the plant was stressed during or after the application.

Since 1987, the trend has been to apply mepiquat chloride in a series of applications each having a lower dose than the single dose application. The first treatment occurs at match head square with a second treatment 7-14 days thereafter. Both treatments are made at a rate within the range from about 0-8 ounces of 4.2 wt % solution per acre with the specific application rate depending on whether the cotton plant was exhibiting signs of being stressed (no application), moderate growth (about 2 ounces of solution per acre), or vigorous growth (about 4 ounces of solution per acre). Thereafter, two additional treatments at 7-14 day intervals may be used with application rates extending up to about 8 ounces of 4.2 wt % mepiquat chloride solution with the specific application rate dependent on the amount of vegetative growth in the field. Further experimentation by individual growers has resulted in a wide variety of multiple application rates.

It would be desirable if the use of mepiquat chloride could be integrated into a system of treatment that would increase plant tissue mass in the roots, stems, and leaves to provide higher levels of nutrient transfer while, at the same time, restricting vegetative growth to enhance fruit production.

The technology of plant herbicides has a continuing desire for enhanced efficacy without a corresponding increase in the application rate. Many herbicides could also use a boost in activity without an increase in the amount of applied herbicide. Farmers and herbicide manufacturers are often faced with a need to control weeds and noxious plants without exceeding the application levels of proven herbicides, if the plants can be controlled at all. Some plants, like Florida Pusley, Bull Grass, Bermuda grass, Dog Fennel, and Primrose are all highly resistant to herbicides proven to be effective.

It would be useful to have a means for increasing the efficacy of agriculturally active ingredients, such as herbicides, without increasing the amount of the applied active ingredient.

SUMMARY OF THE INVENTION

It is an objective of the present invention to provide a composition and method of use therefore to increase the efficacy of agriculturally effective active ingredients.

It is a further objective of the invention to provide a composition and method for its use on fruit-producing plants and seeds that increases the number of fruiting sites on treated plants with the goal of providing increased yields of fruit.

It is another objective of the invention to provide a composition and method for its use in which treated plants grow in a more healthy condition.

In accordance with these and other objectives of the invention that will become apparent from the description herein, a composition according to the invention comprises: (a) an agriculturally effective active ingredient, and (b) an enhancer containing a culture containing bacteria from the genus Bacillus or a soil bacteria in the form of cells, cultures, or suspensions and in an amount sufficient to enhance the effectiveness of said active ingredient. Preferably, the enhancer is free of plant growth hormones when used in combination with plant growth stunting agents, like mepiquat chloride, that suppress plant growth hormones in the treated plant.

Compositions according to the present invention improve the efficacy of the applied agriculturally active ingredient. The same amount of active material that is conventionally applied will be more effective. Lower levels of active ingredient can be used to achieve the same effect as the higher conventional application rate. In addition, plants that have treated with compositions according to the invention are healthier with the attendant benefit of being more resistant to disease or other stress as well as exhibiting higher numbers of fruiting sites and increased yields.

DETAILED DESCRIPTION

The invention provides a method for treating plants with a composition containing an agriculturally effective active ingredient and an enhancer containing a culture of a bacteria selected from the genus Bacillus or a soil bacteria in an amount sufficient to enhance the effectiveness of an agriculturally effective active ingredient applied simultaneously, before, or after application of the enhancer. The increased effectiveness attributable to the bacteria-containing enhancer component can be used to reduce the amount of applied agriculturally effective active ingredient or, when the active ingredient is applied at the same rate, the bacillus increases the effectiveness of the applied agriculturally effective active ingredient. Such increased effectiveness is useful for controlling weeds that are otherwise difficult to control with regular herbicides.

The Bacteria-Containing Enhancer

The enhancer component contains spores, cultures, and suspensions of a bacteria from the genus Bacillus or a soil bacteria. Preferably, the bacteria for the enhancer component is in the form of spores as a result of a suitable adjustment in temperature, pH, salinity, etc.

Suitable bacteria for use in the present invention include those bacteria that exhibit an ability to increase the effectiveness of an agriculturally effective active ingredient by any mechanism. Methods for screening bacterial strains for bioactivity and therefore their capacity to enhance the effectiveness of a plant growth regulator, a herbicide, a systemic fungicide, or a systemic insecticide will be apparent to one of ordinary skill in the art in view of the disclosure and examples set forth herein. For example, a candidate bacterial strain, such as a Bacillus cereus, can be cultured and maintained under standard laboratory conditions. (See e.g., Sambrook et al. “Molecular Cloning: A Laboratory Manual” Cold Spring Harbor Laboratory press (1989)). Specific plants can be chosen for their susceptibility or resistance to a particular agriculturally effective compound such as a herbicide or a plant growth regulator. For example, and as set forth in the Example section contained herein, cotton can be used to evaluate the bioactivity of a bacterial strain in combination with a plant growth regulator as reflected in the number of fruiting sites and/or the number of bolls produced and compared to treating similar plants with only the plant growth regulator or the bacteria. Alternatively, plants susceptible to certain fungal diseases, such as tomato leaf blight, potato scab, wheat stem rust, corn smut, or leaf rust of various plant species, can be grown under controlled conditions and treated either with an appropriate fungicide, a fungicide with a candidate bacteria species, or the bacterial species alone, either before, concurrently, or subsequent to controlled inoculation of the plants with the disease causal organism, to evaluate the capacity of the bacterial species to enhance the activity of the fungicide. These types of experiments can be carried out in the field, under semi-controlled conditions such as a greenhouse, or under relatively controlled conditions such as within a growth chamber. These experiments involve routine screening of organisms for their enhancement effect with compounds or compositions on known plant species, and the parameters used to evaluate the effects are likewise known and routinely measured.

These similar types of screens can be utilized with bacterial strains described herein to provide an enhancement effect to a particular agriculturally effective active ingredient, i.e., the Bacillus cereus strain deposited with the American Type Culture Collection as ATCC 55675 providing an enhancement effect to the plant growth regulator mepiquat chloride and the herbicide atrizine, to screen the bacterial species for potential enhancement with other agriculturally effective active ingredients, such as systemic insecticides and systemic fungicides.

Particularly preferred are cultures, spores, and suspensions of soil bacteria encountered between and among the roots of thriving growths of the plant type in the local soil. Foliar application of the local soil bacteria at an application rate within the range from about 0.1×10¹⁰ CFU/acre to about 10×10¹⁰ CFU/acre enhances the natural mechanisms associated with plant growth and propagation to a level sufficient that the agriculturally active ingredient exhibits enhanced activity on or within the treated plant.

A wide variety of bacterial species within the genus Bacillus and within the known species of soil bacteria are useful within the present invention. The following is a list of species for the present invention:

Bacteria from the genus Bacillus

Bacillus acidocaldarius

Bacillus acidoterrestris

Bacillus alcalophilus

Bacillus alvei

Bacillus aminoglucosidicus

Bacillus aminovorans

Bacillus amyloliquefaciens

Bacillus amylolyticus

Bacillus anthracis

Bacillus aneurinolyticus

Bacillus apiarius

Bacillus azotofixans

Bacillus brevis

Bacillus badius

Bacillus capitovalis

Bacillus cereus

Bacillus circulans

Bacillus cirroflagellosus

Bacillus coagulans

Bacillus colofoetidus

Bacillus cycloheptanicus

Bacillus epiphytus

Bacillus fastidiosus

Bacillus filicolonicus

Bacillus firmus

Bacillus freudenreidii

Bacillus fructosus

Bacillus globigii

Bacillus globisporus

Bacillus insolitus

Bacillus laevolacticus

Bacillus larvae

Bacillus laterosporus

Bacillus lautus

Bacillus lentimorbus

Bacillus lentus

Bacillus licheniformis

Bacillus macerans

Bacillus macquariensis

Bacillus maroccanus

Bacillus macroides

Bacillus medusa

Bacillus megaterium

Bacillus mycoides

Bacillus natto

Bacillus nigrificans

Bacillus pabuli

Bacillus pacificus

Bacillus pantothenticus

Bacillus parabrevis

Bacillus pasteurii

Bacillus polymyxa

Bacillus popilliae

Bacillus pulvifa

Bacillus pulvifaciens

Bacillus pumilus

Bacillus prodigiosus

Bacillus psychrophilus

Bacillus psychrosaccharolyticus

Bacillus racemilacticus

Bacillus sphaericus

Bacillus stearothermophilus

Bacillus subtilis

Bacillus thermodenitrificans

Bacillus thiaminolyticus

Bacillus thuringiensis

Bacillus uniflagellatus

Bacillus validus

Soil Bacteria

Achromobacter pestifer

Alcaligenes eutrophus

Alcaligenes latus

Amycolata autotrophica

Archangium gephyra

Arthrobacter viscosus

Azotobacter chroococcum

Bacillus acidovorans

Bacillus brevis

Bacillus cereus

Bacillus circulans

Bacillus insolitus

Bacillus laterosporus

Bacillus megaterium

Bacillus mojavensis

Bacillus mycoides

Bacillus pasteurii

Bacillus polymyxa

Bacillus psychrosaccharolyticus

Bacillus sphaericus

Bacillus subtilis

Bacillus viscosus

Chainia hygroatrocyanea

Clostridium absonum

Cystobacter fuscus

Cytophaga johnsonae

Ensifer adhaerens

Hyphomicrobium facilis

Microccus luteus

Micromonospora chalcea

Micromonospora coerulea

Micromonospora grisea

Micromonospora polytrota

Microtetraspora glauca

Mycobacterium agri

Mycobacterium aichiense

Mycobacterium aurum

Mycobacterium chitae

Mycobacterium chubuense

Mycobacterium diernhoferi

Mycobacterium fortuitum

Mycobacterium neoaurum

Mycobacterium parafortuitum

Mycobacterium terrae

Mycobacterium thermoresistibile

Myxococcus coralloides

Myxococcus fulvus

Myxococcus macrosporus

Myxococcus xanthus

Nannocystis exedens

Nitrosolobus multiformis

Nocardia brasiliensis

Nocardioides albus

Ochrobactrum anthropi

Polyangium cellulosum

Pseudomonas aeruginosa

Pseudomonas fluorescens

Pseudomonas glathei

Rahnella aquatilis

Saccharobacterium acuminatum

Saccharomonospora viridis

Serratia marcescens

Streptomyces anandii

Streptomyces aureofaciens

Streptomyces chartreusis

Streptomyces cyaneus

Streptomyces cymarogenes

Streptomyces diastatochromogenes

Streptomyces flavogriseus

Streptomyces gelaticus

Streptomyces hygroscopicus

Streptomyces indigocolor

Streptomyces katrae

Streptomyces lipmanii

Streptomyces longisporus

Streptomyces massasporeus

Streptomyces nobilis

Streptomyces odorifer

Streptomyces omiyaensis

Streptomyces parvulus

Streptomyces phaeochromogenes

Streptomyces pseudogriseolus

Streptomyces roseoflavus

Streptomyces rubiginosohelvolus

Streptomyces rutgersensis

Streptomyces sclerogranulatus

Streptomyces toxytricini

Streptomyces violaceoruber

Streptomyces violaceus

Streptomyces violarius

Thermoactinomyces vulgaris

Thiobacillus denitrificans

Thiobacillus thioparus

non-fluorescent Pseudomonas

Rhizobium

Agrobacterium

Corynebacterium ureafaciens

Arthrobacter ureafaciens

Pseudomonas aeruginosa

Bacillus fastidosus

Micrococcus dentrificans

Mycobacterium phlei

Aerobacter aerogenes

Fusarium moniliforme

Histoplasma capsulata

Penicillinum chyrsogenum

Particularly useful are species of B. subtilis, B. cereus, and B. megaterium. Bacillus subtilis and B. cereus are naturally occurring soil saprophytes found throughout the world. In the 1992 edition of the American Type Culture Collection, 182 different strains of B. subtilis are listed and incorporated herein by reference. The following is a list of B. subtilis that would be useful in the present invention:

B. subtilis

ATCC 10783

ATCC 15818

ATCC 15819

ATCC 15245 (Bacillus natto)

ATCC 15134 (Bacillus uniflagellatus)

ATCC 13542

ATCC 13472

ATCC 15575

ATCC 27505

ATCC 21697 (Achromobacter nitriloclastes)

ATCC 15811

ATCC 4925 (Bacillus nigrificans)

ATCC 27370

ATCC 6051a

ATCC 7003

ATCC 11838

ATCC 15563

ATCC 33234

ATCC 25369

ATCC 27689

ATCC 55033

ATCC 13933

ATCC 15244

ATCC 27328

ATCC 12695

ATCC 12100

ATCC 21554

ATCC 21555

ATCC 15561

ATCC 15562

ATCC 9799

ATCC 12711

ATCC 14593

ATCC 4944

ATCC 31002

ATCC 31004

ATCC 9943

ATCC 13407

ATCC 7067

ATCC 29056

ATCC 31524

ATCC 31526

ATCC 21359

ATCC 21360

ATCC 13954

ATCC 13955

ATCC 15044

ATCC 33677

ATCC 31003

ATCC 31522

ATCC 465 N.

ATCC 12432

ATCC 43223

ATCC 13952

ATCC 13953

ATCC 14662

ATCC 15039

ATCC 15040

ATCC 15041

ATCC 15042

ATCC 15043

ATCC 15181

ATCC 15182

ATCC 15183

ATCC 15184

ATCC 21183

ATCC 21336

ATCC 49343

ATCC 6537

ATCC 21394

ATCC 8473

ATCC 31523

ATCC 31525

ATCC 31527

ATCC 29233

ATCC 14660

ATCC 14661

ATCC 31268

ATCC 4925

ATCC 55405

ATCC 9524

ATCC 15476

ATCC 23858

ATCC 23859

ATCC 7060

ATCC 7058

ATCC 7059

ATCC 7480 (Bacillus endoparasiticus).

ATCC 21584

ATCC 31022

ATCC 21331

ATCC 21332

ATCC 21777

ATCC 21778

ATCC 6598 (Bacillus licheniformis)

ATCC 49822

ATCC 23857

ATCC 19221

ATCC 9858

ATCC 21742

ATCC 4529

ATCC 35148

ATCC 33608

ATCC 19549

ATCC 19550

ATCC 21556

ATCC 31340

ATCC 49760

ATCC 53325

ATCC 14807

ATCC 21228

ATCC 15512

ATCC 15841

ATCC 10774

ATCC 31091

ATCC 31092

ATCC 31094

ATCC 31096

ATCC 31097

ATCC 39546

ATCC 39374

ATCC 11774

ATCC 15116

ATCC 35021

ATCC 31954

ATCC 19062

ATCC 23059

ATCC 53115

ATCC 15115

ATCC 13956

ATCC 21952

ATCC 82

ATCC 21603

ATCC 31785

ATCC 21697

ATCC 15477

ATCC 31098

ATCC 19162

ATCC 14617

ATCC 14618

ATCC 33713

ATCC 33714

ATCC 55422

ATCC 6461

ATCC 21007

ATCC 21770

ATCC 6984

ATCC 19163

ATCC 21663

ATCC 19217

ATCC 19219

ATCC 19220

ATCC 21005

ATCC 21006

A preferred B. subtilis strain for use in the present invention includes GB03. Previously, B. subtilis GBO3 was recognized as a biological fungicide and commercially used as a seed treatment under the names KODIAK™ HB or GUS 2000™ by Gustafson, Inc., Plano, Tex. 75093 (EPA Reg. No. 7501-146). This product is available as a 2.75% powder formulation containing not less than 5.5×10¹⁰ viable spores per gram and is to be applied at a rate ranging from 2-4 ounces per 100 pounds of seed. The use directions indicate that the product is to be used for treatment of crop seeds only. This strain is said to colonize the developing root systems and compete with disease organisms that would attack the roots. Foliar application is not listed.

The following is a list of B. cereus that would be useful in the present invention.

B. cereus

ATCC 55675 (BP01)

ATCC 13824 NCIB 2600 (Bacillus cereus var. fluorescens)

ATCC 14603

ATCC 15816

ATCC 15817

ATCC 13472

ATCC 14737

ATCC 9592 (Bacillus metiens).

ATCC 27877

ATCC 19637

ATCC 11950

ATCC 23261

ATCC 6464

ATCC 11773 (Erwinia atroseptica).

ATCC 10876

ATCC 10987

ATCC 4342 (Bacillus lacticola).

ATCC 21182

ATCC 7004 (Bacillus albolactis).

ATCC 10702

ATCC 12480

ATCC 49063

ATCC 2 (Bacillus agri)

ATCC 19265 (Bacillus cereus subsp. alesti)

ATCC 21634

ATCC 12826

ATCC 21768

ATCC 21769

ATCC 21771

ATCC 21772

ATCC 9139

ATCC 21928

ATCC 27522

ATCC 31430

ATCC 43881

ATCC 246 (Bacillus lactimorbus).

ATCC 21770 (Bacillus cereus).

ATCC 21929 (Bacillus cereus).

ATCC 13367

ATCC 31429

ATCC 31293

ATCC 21366 (Bacillus coagulans)

ATCC 25621 (Bacillus medusa)

ATCC 7039 (Bacillus metiens)

ATCC 14893 (Bacillus subtilis).

ATCC 9818 (Bacillus lactis)

ATCC 33018

ATCC 33019

ATCC 55055

ATCC 53522

ATCC 13366

ATCC 7064 (Bacillus siamensis)

ATCC 11949

ATCC 10792 (Bacillus cereus)

ATCC 27348

ATCC 23260 (Bacillus endorhythmos)

ATCC 13061

ATCC 13062 (Bacillus megaterium)

ATCC 25972

ATCC 14579

ATCC 19646

ATCC 49064

ATCC 11778

ATCC 39152

ATCC 19146 FDA Strain PCI 818 (Pseudomonas sp.)

A preferred B. cereus strain for use in the present invention includes strain ATCC No. 55675. In another embodiment, the B. cereus strain for use in the present invention has the characteristics as ATCC No. 55675. In yet another embodiment, the B. substilis strain for use in the present invention has the characteristics as ATCC No. 55675.

The following is a list of B. megaterium that would be useful in the present inventions:

B. Megaterium

ATCC 33166

ATCC 33167

ATCC 33168

ATCC 33169

ATCC 12872

ATCC 33164

ATCC 11478

ATCC 33165

ATCC 11561

ATCC 11561a

ATCC 11561b

ATCC 11561c

ATCC 11561d

ATCC 11561e

ATCC 6458

ATCC 6459

ATCC 14946

ATCC 27327

ATCC 33729

ATCC 9885

ATCC 13639

ATCC 13632

ATCC 15374

ATCC 49099

ATCC 49096

ATCC 43725

ATCC 19136

ATCC 25848

ATCC 4531

ATCC 13402

ATCC 15046

ATCC 15047

ATCC 25300

ATCC 19390

ATCC 13368

ATCC 21181

ATCC 35985

ATCC 39383

ATCC 11562

ATCC 15127

ATCC 15128

ATCC 7703

ATCC 14945

ATCC 15177

ATCC 19218

ATCC 25833

ATCC 15450

ATCC 19135

ATCC 19137

ATCC 21916

ATCC 8245

ATCC 15781

ATCC 31294

ATCC 15117

ATCC 15118

ATCC 19160

ATCC 19161

ATCC 89

ATCC 21209

ATCC 35076

ATCC 49098

ATCC 39118

ATCC 7051

ATCC 7052

ATCC 10778

ATCC 8011

ATCC 7056

ATCC 14581

ATCC 13062

ATCC 49097

ATCC 19213

ATCC 71

ATCC 35075

ATCC 21603

ATCC 21738

ATCC 72

ATCC 21737

ATCC 49095

ATCC 15451

The Active Ingredients

Agriculturally effective ingredients used in the present invention can be selected from a wide variety of materials that act on and through the metabolism of the treated plants. For premixed materials, it is desirable to select active ingredients that do not degrade or otherwise become impaired by extended storage at the conditions used to maintain the bacteria-containing enhancer component in a spore form.

The general term “plant growth regulating agent” encompasses a number of active ingredients that affect a plant in different ways. Generally speaking, plant growth regulators encompass plant growth stunting agents, plant growth enhancing agents, and herbicides.

Suitable plant growth enhancing agents for the present invention include plant growth hormones such as at least one of the 84 identified gibberillins with GA₃, GA₄, GA₅, GA₇ and GA₉ being preferred; cytokinins (e.g., zeatin, kinetin, benzyladenine, dihydrozeatin, and isopentenyl adenine); auxins (e.g., indolacetic acid (IAA), indolebutyric acid (IBA), and naphthalenacetic acid (NAA)); sodium ortho-nitrophenolate; sodium para-nitrophenolate; sodium 5-nitro-guaicolate; polyhydroxycarboxylic acids of 2, 4, 5, and 6 carbon structures; ethephon; and a variety of nitrogen or phosphorous-containing fertilizers.

Suitable plant growth stunting agents useful in the invention include chlormequat chloride, mepiquat chloride, as well as maleic hydrazide and its esters. Such plant growth regulators affect and alter plant metabolic processes to enhance or retard plant growth. All such agents can be used according to the application rates and timing specified by the manufacturer on the product label.

Herbicides include the triazines (e.g., atrazine), the ureas, glyphosate, sulfosate, glyfosinate, and sethoxydim.

Suitable systemic agents that will benefit from enhanced plant uptake, transport, and process assimilation include the systemic pesticides and systemic fungicides. Systemic agents for plants that benefit from the present invention include, inter alia, the insecticides aldicarb, acephate, carbofuran, dimethoate, phorate, and terbufos.

Systemic fungicides that will benefit from the mixtures of the invention include tridemorph, metalaxyl, iprodione, fosetyl-aluminum, thiophanate, benomyl, triadimefon, carboxin, oxycarboxin, carbendazim, thiabendazole, thiophanate, ethirimol, bupirimate, and dimethirimol.

Plants that can be treated by the present invention include virtually any plant grown in soil and that is affected by an agriculturally effective active ingredient. Exemplary plants include commodity grain crops (e.g., corn, wheat, and soybeans), sorghum, desired and undesired grasses, weeds, herbs, etc.

The invention is well suited to increased production of fruit in plants that produce fruiting sites from which fruit will grow. Such plants preferably include any of the raw agricultural commodity and especially cotton, soybeans, peanuts, grapes, apples, citrus (e.g., lemons, limes, oranges, grapefruit), berries (e.g., strawberries, blackberries, raspberries), tubers (e.g., potatoes, sweet potatoes), corn, cereal grains (e.g., wheat, rice, rye), tomatoes, onions, cucurbits (e.g., watermelon, cucumbers, and cantaloupes).

Method of Use

The compositions of the present invention may take the physical form of a liquid, emulsion, suspension, solid granule, aggregate, or composite granule (e.g., active ingredient solids carried on an inert carrier particle). Application of each physical form to plant foliage will generally proceed with conventional techniques.

Gram positive bacteria strains can be used in the enhancer component in the form of cells, spores, cultures, or suspensions thereof. In a liquid or dispersible solid forms, the enhancer is added to a spray tank or other form of liquid distribution reservoir as a stable, aqueous concentrate solution exhibiting an equivalent spore concentration within the range from about 300,000 colony forming units per milliliter (CFU/ml) to about 1.5 million CFU/ml, preferably about 1 million to about 1.2 million CFU/ml to make a composition that is applied to plant foliage at a rate within the range from about 0.1×10¹⁰ CFU/acre to about 100×10¹⁰ CFU/acre, preferably at a rate within the range from about 0.1×10¹⁰ CFU/acre to about 10×10¹⁰ CFU/acre, and most preferably within the range from about 0.5×10¹⁰ CFU/acre (0.5 fl. oz./acre of concentrate) to about 8×10¹⁰ CFU/acre (2 fl. oz./acre of concentrate). Optionally and in a preferred embodiment, the spray tank will also contain the agriculturally effective active ingredient component for simultaneous application of both components.

Solid forms of the components can be dry mixed or formed into aggregates before broadcast. One or more of the conventional adjuvants may be used to enhance dispersion, breakdown, adhesion to foliage, etc.

The specific application rate can vary somewhat depending on the method by which the solution is to be applied to the plant surfaces. For example, aerial spraying will employ a different dilution rate and application quantity than boom spraying, manual sprayers, or broadcast of granules. Conventional equipment can be used for the application. If desired, the enhancer component can be mixed with other treatments and applied simultaneously or can be applied in a discrete treatment step. Foliar application is the preferred method for increasing the number of fruiting sites on fruit-producing plants.

The concentrate can also be used to formulate a ready-to-use, packaged mixture. So prepared, the enhancer is diluted to an amount in the package container that is within the range from about 150,000 CFU/ml to about 600,000 CFU/ml and with conditions adequate to ensure that the bacterial component remains in a spore form but will become vegetative after application.

For many bacteria, use of a pH of less than 7 (i.e., acidic) will maintain the bacteria in a spore form. If necessary, any of the conventional acidifying agents or buffers (preferably food grade or those classified as “Generally Regarded As Safe” by the U.S. Environmental Protection Agency) may be used to maintain a suitable acidic pH to ensure storage stability. Under such acidic conditions, the spores remain stable and exhibit good storage stability. When diluted for use and following application, the pH of the solution will raise to greater than 7 thereby causing the bacteria in the enhancer to become live, vegetative colonies. The bacteria will thereby reproduce on the treated plant surfaces and facilitate or translocation of the agriculturally effective active ingredient.

The bacteria-containing enhancer component can be applied as a discrete treatment or simultaneously with a variety of other agriculturally effective active ingredients. Useful agriculturally effective active ingredients include plant growth enhancing agents, plant growth stunting agents, herbicides, systemic insecticides, and systemic fungicides. Preferably, the composition is a combination of either a plant growth stunting agent or herbicide and an enhancer containing a substantially pure strain of B. subtilis, B. cereus, or ATCC 55675 (BP01) applied at the rate of at least 0.1×10¹⁰ CFU/acre.

In a particularly preferred embodiment, a gibberellin-free enhancer containing the bacillus is applied to the foliage of cotton plants at the same time the plants are treated with mepiquat chloride. An aqueous tank mixture containing the bacteria-containing enhancer (preferably ATCC 55675) and mepiquat chloride is a convenient method for simultaneously applying the components. If premixed, the mepiquat achloride and bacteria-containing enhancer can be stored readily at a pH within the range from about 4-6.5, most preferably within the range of about 5-6.5.

It should be noted that formulations according to the present invention desirably do not include combinations of materials that attempt to act in a contradictory fashion on the plant metabolism. For example, mepiquat chloride is commonly used on cotton foliage to suppress plant growth hormones and stunt the vegetative growth of the plant. A formulation would preferably not be prepared that included plant growth hormones because the effects of the mepiquat chloride and the growth hormones would place inconsistent demands on the plant metabolism, reduce the efficacy of the mepiquat chloride, and lead to inconsistent results. With the present invention, however, the combination of mepiquat chloride and ATCC 55675 consistently produces treated plants that have higher yield, more healthy growth, and a higher resistance to disease.

While not wishing to be bound by any particular theory of operation and with respect to the combined use of the bacteria-containing enhancer and mepiquat chloride on cotton, the bacteria appears to be affecting the plant growth mechanism to increase the retention of bolls on fruiting sites 1 and 2 and increasing the number of bolls overall by producing and retaining fruiting sites on normally vegetative branches.

For the present invention, mepiquat chloride is used at the application rates and during the conventional stages of cotton plant growth. Conventionally applied rates of mepiquat chloride are up to about 60 g/acre (25 g/acre) or about 1-16 ounces per acre with individual application rates falling within the range from about 2.5 g/acre (1.0 g/hectare) for a 2 ounce/acre application of 4.2 wt % solution to 10 g/acre (4.1 g/hectare) for an 8 ounce/acre application of the same 4.2 wt % solution. If mepiquat chloride of higher or lower purity and/or activity is used, the specific application rate should be adjusted up or down according to the change in conventional mepiquat chloride activity.

EXAMPLES Example 1

An aqueous mixture of 4.2 wt % mepiquat chloride and 560,000 CFU/ml B. cereus (lab sample BP01, ATCC 55675) was prepared in a 1:1 volumetric ratio. This solution was applied by conventional sprayer in four applications to the foliage of growing cotton plants in test areas of fields A (10 of 50 acres in Mississippi) and B (8 of 60 acres in Tennessee). Field A was treated with one dose at the range of 4 fluid ounces per acre followed in five weeks by a treatment at the rate of 8 fluid ounces per acre.

Field B received a different treatment regiment. The first three applications in Field B were at the rate of 4 fluid ounces per acre. (The first and second were 24 days apart. The second and third were at 6 days apart). The final application in Field B was at the rate of 8 fluid ounces per acre 11 days later. In all cases, the control fields were treated with the same rate of only mepiquat chloride.

After treatment, sampling in Fields A and B was conducted by hand of representative rows. All known biases were placed in favor of the control treatment with only mepiquat chloride.

In Fields A and B, beginning and final plant heights of the treated cotton plants was measured. The total plant heights of the control fields was comparable to those treated according to the invention. See, Table 1.

TABLE 1 Field Beginning Height (in.) Final height (in.) A - Control 293 601 A - Treated 291 597 B - Control 338 580 B - Treated 334 636

During the growing season, the differences between the control and treated plants were readily observed. When the young plants contained only squares in the first month of fruiting, an unusually high number and size of additional fruiting branches were seen where the primary fruiting branch exits the main stem. Many were as large or almost as large as the primary fruiting branch and contained fruit prior to first bloom. Extra fruit was also observed at the intersection of the fruiting branches and main stem.

As the bloom extended up the plant and only bolls or missing sites were found below the bloom, an unexpected amount of back fruiting was also observed. Double fruiting was observed in squares, blooms, small bolls, and “now open bolls” (rare). Although double fruiting occurred in both the control and treated plants, the instances were higher in the treated plants.

About 3-4 weeks before the end of the growing period, the plants were inspected for the number of fruiting sites, the number of bolls on nodes 1 and 2, as well as the type of boll. Table 2 reports the results of that inspection.

TABLE 2 Open Open + Other Field Sites Bolls % Bolls Green Bolls A - Control 240 115 48  73 270 25 A - Treated 292 155 53 173 359 103 B - Control 344 207 60 — — 81 B - Treated 366 234 64 — — 163

In Field B and at the end of the test period, 20 plants were inspected. The plants were at cut-out. There were no open bolls in the control group and only 7 in the treated group. The younger fruit, however, were larger and older for the treated plants compared to the control plants. This indicates that the treated plants were growing at a faster and more favorable rate. The treated plants had a total of 265 bolls compared to 238 bolls for the control group, an improvement of 11%.

The treated plants also exhibited an increase in the number of fruit on the vegetable branches in the ratio of 86:50 (72% increase). The treated plants also produced an increase in other fruit, i.e., those on fruiting branch positions 3 and wider, at the ratio of 86:50 (72% increase). The estimated weight (seed cotton) of the fruit from the 20 plants was also higher in the treated plants, 1796 g. v. 1456 g. (23% increase).

The extra fruit on the plants brought an expectation that the additional plant parts and young fruit would be a drain on the plant's system. Bolls would open sooner, but would adversely affect yield. This expectation was not realized. The treated plants grew at a faster rate and in a more healthy condition than the control.

To reduce bias, all link was then harvested by hand from the test fields including unfluffed lint from partially opened bolls. Such unfluffed lint are usually from bolls that never opened correctly and are low on the plant or from bolls just opening that are high on the plant. Table 3 reports the weight of seed cotton and the number of green bolls per 10 foot of row in the treated and control fields.

TABLE 3 Field Seed cotton (g.) Number of Green Bolls A - Control 5322 97 A - Treated 6287 0 B - Control 4532 175 B - Treated 5058 42

The test results show that the combination of mepiquat chloride applied simultaneously with an enhancer containing ATCC 55675 according to the invention produces higher cotton yields and healthier plants than use of mepiquat chloride alone.

Example 2

Bacillus cereus strain BP01 (ATCC 55675) was used in combination with a widely used herbicide, atrazine, to determine whether the BP01 would affect control over weeds that are recognized as difficult to fill with triazines. Atrazine is used to provide season-long control in corn, sorghum, and other crops at a suggested rate of 2 pounds active ingredient per acre. At sufficiently high rates, such as those used in this example, atrazine is recognized for its ability to provide nonselective weed control.

In sandy soil, three replicates of each test were performed in 12 ft.×25 ft. plots using a randomized complete block pattern. The weeds were 2-6 inches (5-15 cm) in height at the time of treatment. Atrazine was applied from aqueous solution at rates equivalent to either 1 or 2 pounds of active ingredient per acre. BP01 concentrate was added to the atrazine in an amount equivalent to either 0.5 or 1 fluid ounce per acre (0.5-1×10¹⁰ CFU per acre). For comparison, crop oil concentrate (about 85% paraffinic oil and about 17% surfactant) was used as is conventional with triazine herbicides to increase their efficacy. Tables 4-8 report the degree of control for Florida Pusley (Table 4), Bull Grass (Table 5), Bermuda grass (Table 6), Dog Fennel (Table 7), and Primrose (Table 8).

TABLE 4 Florida Pusley % Control After Treatment Treatment 4 days 9 days 16 days 23 days Control 0 0 0 0 Atrazine (1 lb.) 3 33 42 45 Atrazine (2 lb.) 17 57 72 83 Atrazine (1 lb) + 28 62 68 68 crop oil conc. (1 gal.) Atrazine (1 lb) + 27 43 60 60 0.5 oz. BP01 Atrazine (1 lb) + 22 53 65 67 1 oz. BP01

TABLE 5 Bull Grass % Control After Treatment Treatment 4 days 9 days 16 days 23 days Control 0 0 0 0 Atrazine (1 lb.) 10 40 40 55 Atrazine (2 lb.) 25 70 80 90 Atrazine (1 lb) + 25 40 65 60 crop oil conc. (1 gal.) Atrazine (1 lb) + 35 40 55 63 0.5 oz. BP01 Atrazine (1 lb) + 30 60 80 90 1 oz. BP01

TABLE 6 Bermuda grass % Control After Treatment Treatment 4 days 9 days 16 days 23 days Control 0 0 0 0 Atrazine (1 lb.) 0 10 15 15 Atrazine (2 lb.) 8 15 22 27 Atrazine (1 lb) + 25 27 30 43 crop oil conc. (1 gal.) Atrazine (1 lb) + 7 20 37 68 0.5 oz. BP01 Atrazine (1 lb) + 10 23 53 63 1 oz. BP01

TABLE 7 Dog Fennel % Control After Treatment Treatment 4 days 9 days 16 days 23 days Control 0 0 0 0 Atrazine (1 lb.) 15 40 55 60 Atrazine (2 lb.) 17 55 70 95 Atrazine (1 lb) + 25 75 93 98 crop oil conc. (1 gal.) Atrazine (1 lb) + 33 88 96 99 0.5 oz. BP01 Atrazine (1 lb) + 30 90 97 100 1 oz. BP01

TABLE 8 Primrose % Control After Treatment Treatment 4 days 9 days 16 days 23 days Control 0 0 0 0 Atrazine (1 lb.) 10 40 50 60 Atrazine (2 lb.) 30 65 70 80 Atrazine (1 lb) + 20 83 88 93 crop oil conc. (1 gal.) Atrazine (1 lb) + 20 60 72 88 0.5 oz. BP01 Atrazine (1 lb) + 23 70 65 80 1 oz. BP01

From Tables 4-8, it can be seen that BP01 generally improve the effectiveness of the atrazine at 23 days after treatment. The control rate at 1 lb. of atrazine with the bacillus was better than the control rate of 2 lb. atrazine for Bermuda grass, Dog Fennel, and Primrose, and the two treatments has the same control rate for Bull Grass. Only with Florida Pusley and 1 lb/acre of atrazine with BP01 was the control rate reduced relative to the 2 lb/acre treatment with atrazine.

Similarly, the BP01 also improved the control rate of atrazine relative to a mixture of atrazine and crop oil concentrate in all weeds except for Florida Pusley and Primrose. Such an improvement suggests that the bacillus is not acting as a surfactant, but is enhancing effectiveness by either or both of the metabolic activity or translocation characteristics of the co-applied agent.

The preceding are intended solely for purposes of illustrating the invention and are not intended to act as limitations on the scope of the appended claims. 

What is claimed is:
 1. A method for enhancing the effectiveness of a herbicide by applying to a plant (a) an agriculturally effective active ingredient of a plant growth stunting agent or herbicide; and (b) an enhancer selected from the group consisting of spores, cultures, or suspensions of a suitable Bacillus or soil bacteria.
 2. The method of claim 1 wherein siad agriculturally effective active ingredient and said enhancer are applied in a composition.
 3. The method according to claim 1 wherein said agriculturally effective active ingredient is a triazine, glyphosate, or sulfosate.
 4. The method of claim 1 wherein said enhancer contains a strain of bacteria from the genus Bacillus.
 5. The method of claim 1 wherein said enhancer contains a strain of soil bacteria.
 6. The method of claim 1 wherein said enhancer contains a B. cereus.
 7. The method of claim 1 wherein said enhancer contains a B. cereus having a characteristic of ATCC
 55675. 8. The method of claim 1 wherein said enhancer contains a B. subtilis.
 9. The method of claim 1 wherein said enhancer contains a B. subtilis having a characteristic of ATCC
 55675. 10. The method of claim 1 wherein said enhancer contains B. megaterium.
 11. The method of claim 1 wherein said enhancer contains ATCC
 55675. 12. A composition of enhancing the activity of a plant growth regulating agent comprising a suitable Bacillus, spore, culture and suspension thereof and a plant growth stunting agent, wherein the composition does not contain ATCC
 55675. 13. The composition of claim 12 wherein said plant growth regulating agent comprises mepiquat chloride.
 14. The composition of claim 12 wherein said plant growth regulating agent comprises chlorinequat chloride.
 15. The composition of claim 12 wherein the Bacillus is a Bacillus cereus.
 16. The composition of claim 12 wherein the Bacillus has a characteristic of ATCC
 55675. 17. The composition of claim 12 wherein the Bacillus is a Bacillus subtilis.
 18. A composition for enhancing the activity of an agriculturally effective active ingredient comprising an aqueous mixture comprising: (a) an agriculturally effective active ingredient selected from the group consisting of a plant growth regulating agent, herbicide, systemic fungicide, and a systemic insecticide; and (b) an enhancer selected from the group consisting of spores, cultures, or suspensions of a suitable Bacillus or soil bacteria at a pH sufficiently less than 7 to maintain said enhancer in spore form, provided that the composition does not contain plant growth hormones when said plant growth regulating agent is a plant growth stunting agent.
 19. The composition of claim 18 wherein said plant growth regulating agent comprises mepiquat chloride, chlormequat chloride, or ethephon.
 20. The composition of claim 18 wherein said agriculturally effective active ingredient comprises a herbicide.
 21. The composition of claim 20 wherein the herbicide is a triazine, glyphosate, or sulfosate.
 22. The composition of claim 18 wherein said enhancer contains a strain of bacteria from the genus Bacillus.
 23. The composition of claim 18 wherein said enhancer contains a strain of soil bacteria.
 24. The composition of claim 18 wherein said enhancer contains a B. cereus.
 25. The composition of claim 18 wherein said enhancer contains a B. cereus having a characteristic of ATCC
 55675. 26. The composition of claim 18 wherein said enhancer contains a B. subtilis.
 27. The composition of claim 18 wherein said enhancer contains a B. subtilis having a characteristic of ATCC
 55675. 28. The composition of claim 18 wherein said enhancer contains B. megaterium.
 29. The composition of claim 18 wherein said enhancer contains ATCC
 55675. 30. A nonliquid composition comprising: a nonliquid mixture of (a) an agriculturally effective active ingredient selected from the group consisting of a plant growth stunting agent, ethephon, a plant growth hormone, naphthalenacetic acid, sodium ortho-nitrophenolate, sodium para-nitrophenolate, sodium 5-nitro-guaicolate, polyhydroxycarboxylic acids of 2, 4, 5, and 6 carbon structures, and a herbicide; and (b) an enhancer containing spores or cultures of a suitable Bacillus or soil bacteria, provided that the composition does not contain plant growth hormones when a plant growth stunting agent is the active ingredient.
 31. The composition of claim 30 wherein said active ingredient is mepiquat chloride, chlormequat chloride, or ethephon.
 32. The composition of claim 30 wherein said herbicide is glyphosate or sulfosate.
 33. A method for increasing the number of fruiting sites on a fruiting plant by a. diluting a composition containing (a) an agriculturally effective active ingredient of a plant growth regulating agent; and (b) an enhancer containing spores of a suitable Bacillus or soil bacteria at a pH of less than 7 and sufficiently low to maintain said Bacillus in spore form, and b. applying the diluted composition to foliage of a plant containing fruiting sites.
 34. The method of claim 33 wherein said fruiting plant is cotton.
 35. The method of claim 33 wherein said plant growth regulating agent is a plant growth stunting agent or ethephon.
 36. The method of claim 33 wherein said enhancer contains a strain of bacteria from the genus Bacillus.
 37. The method of claim 33 wherein said enhancer contains a B. cereus having a characteristic of ATCC
 55675. 38. The method of claim 33 wherein said enhancer contains a B. subtilis having a characteristic of ATCC
 55675. 39. The method of claim 33 wherein said enhancer contains a B. megaterium, a B. subtilis, or a B. cereus.
 40. The method of claim 33 wherein said enhancer contains ATCC
 55675. 41. A method for enhancing the effectiveness of an agriculturally effective active ingredient by applying to a plant (a) an agriculturally effective active ingredient of a plant growth stunting agent, ethephon, or a herbicide; and (b) an enhancer selected from the group consisting of spores, cultures, or suspensions of a suitable Bacillus or soil bacteria.
 42. The method of claim 41 wherein said agriculturally effective active ingredient is a triazine, glyphosate, or sulfosate.
 43. The method of claim 41 wherein said active ingredient is mepiquat chloride or chlormequat chloride.
 44. The method of claim 41 wherein said enhancer contains a strain of bacteria from the genus Bacillus.
 45. The method of claim 41 wherein said enhancer contains a strain of soil bacteria.
 46. The method of claim 41 wherein said enhancer contains a B. cereus.
 47. The method of claim 41 wherein said enhancer contains a B. cereus having a characteristic of ATCC
 55675. 48. The method of claim 41 wherein said enhancer contains a B. subtilis.
 49. The method of claim 41 wherein said enhancer contains a B. subtilis having a characteristic of ATCC
 55675. 50. The method of claim 41 wherein said enhancer contains B. megaterium.
 51. The method of claim 41 wherein said enhancer contains ATCC
 55675. 